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					Workshop on Circadian Clocks in Plants and Fungi
Mathematical Biosciences Institute
Ohio State University, Columbus
October 24-29, 2010


 Circadian Control at the
Post-Transcriptional Level:
  the Gonyaulax Rhythms
                  Woody Hastings
     Department of Molecular & Cellular Biology
                Harvard University
              Cambridge, MA 02138
  Gonyaulax – a dinoflagellate
• Several different daily rhythms measured
• Luminescence, cell division, aggregation,
  photosynthesis, protein abundance
• Light emission as a biochemical marker for
  a rhythm: genes, proteins
• Luciferase enzyme correlates with rhythm
NIGHT HAULING by ANDREW WYETH, showing
   DINOFLAGELLATE BIOLUMINESCENCE
DINOFLAGELLATE LUMINESCENCE in BREAKING WAVES
                             La Jolla CA, David Welsh
     MECHANICAL STIMULATION ELICITS
DINOFLAGELLATE BIOLUMINESCENCE in FLASK
ENTRAINED & FREE RUNNING RHYTHM
 OF LUMINESCENCE IN GONYAULAX



                                             Absence of
                                             damping?




                          Hastings, J.W. (1960) Cold Spring
                          Har Symp Quant Biol 25: 131-144
     Some Gonyaulax results that can
        be discussed for modeling
• Translational control of circadian protein
  synthesis
• Temperature compensation: Q10 less than 1
• Reversible loss of rhythms at a low temperature
• Different rhythms may have different periods
• Evidence for communication of circadian phase
  via the medium; synchronization
• Infradian and ultradian rhythms: a dinoflagellate
  circannual rhythm
Gonyaulax CELL SHOWING SCINTILLONS by FLUORESCENCE
                    (Carl H. Johnson)
    Gonyaulax CELLS at NIGHT (left) & DAY PHASES
FLUORESCENCE of LUCIFERIN in SCINTILLONS (Carl H. Johnson)
  Two proteins in Gonyaulax Bioluminescence
             Reaction is pH-dependent
Luciferin binding protein (LBP) & Luciferase (Lase)
GONYAULAX LUCIFERASE ACTIVITY RHYTHM Hastings & Bode, 1963
     Is the rhythm due to the
amount of luciferase or its regulation?
    BOTH LBP and Lase are
      SYNTHESIZED and
    DESTROYED EACH DAY

     CELLULAR CONCENTRATIONS
     EXHIBIT a CIRCADIAN RHYTHM
LUCIFERASE PROTEIN EXHIBITS A CIRCADIAN RHYTHM in LL



                                         Johnson et al.1984
                                         Science 223
WESTERN BLOTS LUCFERIN BINDING PROTEIN, LD & LL
                                  Morse et al., 1989 PNAS 86
       LBP mRNA DOES NOT CYCLE in Gonyaulax
LBP SYNTHESIS & ABUNDANCE are STRONGLY CIRCADIAN
                                    Morse et al., 1989 PNAS 86


                                 LBP mRNA




                                LBP abundance


          LBP synthesis
  SYNTHESIS of MANY PROTEINS is CIRCADIAN CONTROLLED
    in vivo PULSE LABELING    2D gels    MILOS et al, 1989




Circles: day phase                         MILOS ET AL, 1990
synthesis                                  Naturwissenschaften 77:87-89




 Squares: night
 phase synthesis
SYNTHESIS of PROTEINS in vitro is NOT CLOCK CONTROLLED
                                        2D gels

                                      MILOS ET AL, 1990
                                      Naturwissenschaften 77:87-89
   PATTERNS of CLOCK-CONTROLLED PROTEIN SYNTHESIS in Gony
Markovic et al., 1996                     2D gels
J. Biol. Rhythms                          Measurements
                                          every 3 hours




                                           p32 PCP
                                           p33 OEE1
                                           p45 GAPDH
                                           p55 Rubisco II
                                           P75 LBP
mRNA LEVELS ARE CONSTANT in LD & LL
GAPDH SYNTHESIS, ACTIVITY & ABUNDANCE RHYTHMS
                              Fagan, Morse & Hastings, 1999
PROTEIN TURNOVER AFFECTS AMPLITUDE of ABUNDANCE RHYTHM
     Some Gonyaulax results that can
        be discussed for modeling
• Translational control of circadian protein
  synthesis
• Temperature compensation: Q10 less than 1
• Reversible loss of rhythms at a low temperature
• Different rhythms may have different periods
• Evidence for communication of circadian phase
  via the medium; synchronization
• Infradian and ultradian rhythms: a dinoflagellate
  circannual rhythm
EFFECT OF TEMPERATURE ON
RHYTHM OF LUMINESCENCE
Hastings& Sweeney, 1957




Gonyaulax
Polyedra
Q10 less than 1

NOTE 11.5o
Q10 LESS THAN 1 ATTRIBUTED TO OVER-COMPENSATION
     HIGHER TEMPERATURES ALSO DECREASE
AMPLITUDE of LUMINESCENCE RHYTHM in GONYAULAX
AMPLITUDE AND PERIOD BOTH HAVE A Q10 LESS THAN 1.0
LOSS OF RHYTHMICITY BELOW 12O C




                         Hastings & Sweeney, 1957
                         PNAS 43: 804-811 and (1960)
                         Cold Spring Har Symp Quant Biol
     Some Gonyaulax results that can
        be discussed for modeling
• Translational control of circadian protein
  synthesis
• Temperature compensation: Q10 less than 1
• Reversible loss of rhythms at a low temperature
• Different rhythms may have different periods
• Evidence for communication of circadian phase
  via the medium; synchronization
• Infradian and ultradian rhythms: a dinoflagellate
  circannual rhythm
LOW TEMPERATURE for 12 hr STOPS the CLOCK for 12 hr
  Only 1.5° C between permissive and non-permissive
                                           Njus et al. 1977
                                           J Comp Physiol
                                           117: 335-344


             20o


             20o
      RESUMPTION of RHYTHM AFTER LOW TEMP STARTS at CT12
Njus et al. 1977
J Comp Physiol
117: 335-344
• Other conditions that cause loss of
  rhythmicity also resume at CT12 when
  permissive conditions are restored

 Loss of oscillation under non-
permissive conditions (e.g.,low
 temperature) could be due to
  protein subunit dissociation
     Some Gonyaulax results that can
        be discussed for modeling
• Translational control of circadian protein
  synthesis
• Temperature compensation: Q10 less than 1
• Reversible loss of rhythms at a low temperature
• Different rhythms may have different periods
• Evidence for communication of circadian phase
  via the medium; synchronization
• Infradian and ultradian rhythms: a dinoflagellate
  circannual rhythm
    Free-running Human Subject Demonstrating Switch From “Internally
       Synchronized Rhythms” to “Real Internal Desynchronization”
                                Time (hours)
Time (days)




                                               sleep - wake




                               temp




                                      Wever R. Int J Chronobiol 3: 19, 1975
    In Gonyaulax SEVERAL DIFFERENT
 RHYTHMS PEAK at DIFFERENT PHASES
Are THEY CONTROLLED by ONLY a SINGLE
 MECHANISM at the MOLECULAR LEVEL?
Spontaneous flashing and glow
DIFFERENT OSCILLATORS CONTROL GLOW & FLASHING
GONYAULAX INTERNAL DESYNCHRONIZATION OF TWO RHYTHMS
                                            ROENNEBERG
     Some Gonyaulax results that can
        be discussed for modeling
• Translational control of circadian protein
  synthesis
• Temperature compensation: Q10 less than 1
• Reversible loss of rhythms at a low temperature
• Different rhythms may have different periods
• Evidence for communication of circadian phase
  via the medium; synchronization
• Infradian and ultradian rhythms: a dinoflagellate
  circannual rhythm
Only Slight Damping in Cyanobacterial
Gene Expression Rhythm in vivo with
 Bacterial Luciferase as a Reporter
                       12   60    84      108    132       156        180       204
                                                                                             228
               10000
Luminescence




               8000



               6000



               4000



               2000




                       0    48    72      96     120       144       168        192          240

                            Hours in Constant Light
                                                       Kondo, Johnson Golden et al., 1993,
                                                       PNAS
  HUMORAL FACTOR(S)?
• ABSENCE OF DESYNCHRONIZATION
• MIXING OUT-OF-PHASE CULTURES
  SUGGESTED AN INVOLVEMENT
• BRAIN EXTRACTS TESTED FOR
  EFFECT OF Gonyaulax RHYTHM
• RECENTLY, MANY PEPTIDES HAVE
  BEEN ISOLATED FROM SCN
 MIXING TWO OUT-OF-PHASE CULTURES
SEPARATE   MIXED   MIXED, FRESH MEDIUM
                               Broda et al.1985 Cell
                               Biophysics 8: 47-67
BRAIN EXTRACT: EFFECT ON PERIOD
                              Krieger & Hastings
ACTIVE SUBSTANCE is CREATINE: PERIOD APPEARS to be
  RELATED to PHOSPHORYLATION STATUS of PROTEINS




                                              Roenneberg et al.
                                              1988 Nature
EFFECTS OF KINASE INHIBITORS ON PERIOD (TAU)
PERIOD EFFECT OF PROTEIN PHOSPHATASE INHIBITORS
   ULTRADIAN and INFRADIAN
    BIOLOGICAL RHYTHMS

• HOW MIGHT THESE be INCORPORTED into
  MODELS of BIOLOGICAL RHYTHMS MORE
  GENERALLY?
• FREE-RUN in CONSTANT CONDITIONS
• FEW STUDIES of ENTRAINMENT
• TEMPERATURE COMPENSATION not
  TYPICALLY FOUND, but LITTLE STUDIED
ANNUAL RHYTHM: DINOFLAGELLATE CYST GERMINATION
Alexandrium fundyense                   MATRAI, 2005
           BIOLOGICAL LABORATORIES - HARVARD UNIVERSITY




                                                                   Bess & Victoria
                                                                   May 2007
                                                                   75th Birthday




HASTINGS LAB                                          RESEARCH SUPPORT
Thérèse Wilson and many former members, including     NSF, NIH, ONR
Neil Krieger, Laura McMurry, David Njus, Jay Dunlap   Collaborator: Beatrice Sweeney
Carl Johnson, Till Roenneberg, David Morse
RED TIDE, YELLOW SEA




                       10 m
LUCIFERASE has 3 HOMOLOGOUS CATALYTIC DOMAINS




                                       Li et al., 1997
                                       PNAS 94: 8954-cc58
Figure 3

             Luciferin Binding Protein has 4 homologous domains
                      10        20        30       40          50              60
  NsLBP_D1
  NsLBP_D2
  NsLBP_D3
  NsLBP_D4
  LpLBP_D1
  LpLBP_D2
  LpLBP_D3
  LpLBP_D4

                      70        80        90       100         110             120
  NsLBP_D1
  NsLBP_D2
  NsLBP_D3
  NsLBP_D4
  LpLBP_D1
  LpLBP_D2
  LpLBP_D3
  LpLBP_D4
                      130       140       150
  NsLBP_D1
  NsLBP_D2                                               Liu & Hastings 2007
  NsLBP_D3
  NsLBP_D4                                               PNAS 104: 696-703
  LpLBP_D1
  LpLBP_D2
  LpLBP_D3
  LpLBP_D4
GONYAULAX MELATONIN RHYTHM

                    Hardeland, Balzer et al. 1992
PHOTOPERIODIC INDUCTION OF CYSTS IN GONYAULAX
                        Hardeland, Balzer et al. Science 1991
A NOVEL 22-NT SEQUENCE IN THE LBP 3’ UTR
   BINDS A PROTEIN IN CELL EXTRACTS
                            Mittag et al., 1994
                            PNAS 91: 5257-61




   Gel Retardation




                           22 nt
Mittag et al., 1994 PNAS 91: 5257-61
     Circadian Changes of Binding Activities
of CHLAMY 1, 2 and 3 with Gony 22 nt sequence




                                                     RNA alone
         LL 14 18 22 26 30 34 38 42 46 50 54 58 62


                                                                 Gel retardation

     3
     2




     1




                                                                      Mittag, PNAS, 1996
                   CHLAMY1 is a Heterodimeric
                           RNA-Binding Protein

                                             C1- 45 kDa


                                KH      KH     KH    PGG/    WW
                                                     YGG
KH: Lysine homology domain
WW: Protein - protein interaction domain




                                           C3- 52 kDa


                          RRM     RRM         Met-rich    RRM
RRM: RNA recognition motif domain




                                                            Zhao et al., Eukaryot. Cell 2004
POSTULATED CORE CIRCADIAN OSCILLATOR
   WITH INPUT AND OUTPUT PATHWAYS
Silencing or Over expression of C3 Causes a Shift in
         Acrophase of Circadian Phototaxis
                   WT          C3-sil                   WT                  C3-sil
      100           50   25   100 100             100    50 25             100 100

C3                                                                                          C1



              350
              325
              300
              275
                                                                           Wt
              250
                                                                            = 24,7 h
     E (mV)




              225
              200
              175                                                          C3sil-6
              150                                                           = 24,3 h
              125
              100
              75
              50
              25
               0
                         1     2        3    4     5         6         7
                               Days in darkness
                                                                 Iliev, Voytsekh et al., Plant Physiol. 2006
  Overexpression or Silencing of C1 Co-Regulates C3 and
Disturbs the Rhythm, Causing Arrhythmicity in Some Cases

                      WT          C1-ox                      WT                C1-ox

            90         60   30   30    30           90       60       30    30       30

   C1                                                                                         C3



                 225

                 200
                                                                               C1-ox93
                 175                                                           arrhythmic
        E (mV)




                 150
                                                                                WT
                 125                                                            =   24,7 h
                 100

                  75

                  50

                 25

                  0
                            1    2       3      4        5        6        7
                                      Days in darkness
                                                                  Iliev, Voytsekh et al., Plant Physiol. 2006
ABSOLUTE AMOUNTS of SYNTHESIS and ABUNDANCE
               NOT KNOWN
                                   Morse et al., 1989 PNAS 86


                                 LBP mRNA




                               LBP abundance


          LBP synthesis